Various assa methods are currently used for monitoring either urokinase or tissue plasminogen activator, TPA. TPA and urokinase are found in blood and are drugs available for thrombolytic therapy from Genentech, Inc. and Abbott Laboratories, respectively. Both are proteolytic enzymes which convert plasminogen to plasmin, i.e., a serine protease with serine at it active center or site.
Plasmin is responsible for dissolving the fibrin clots or thrombi, formed in various pathologic states such as myocardial infarcts and strokes.
Prior assays for monitoring plasminogen activators such as TPA rely either on reaction of a chromogenic substrate with the plasmin generated in a discontinuous two-stage assay, or a continuous linked assay using a chromogenic substrate with analysis of the parabolic reaction curve of the chromogenic product versus time. The disadvantage of these methods is that the plasmin formed can feed back and cleave either the enzyme urokinase or TPA or the plasminogen substrate resulting in distorted kinetic properties.
Prior methods also include ligand assay using either radioimmunoassay or ELISA, but the disadvantage of this is that it measures the antigenic properties of the plasminogen activator, such as TPA, and thus can not distinguish between active and inactive plasminogen activator, i.e., that which has reacted with inhibitors. Therefore, such assay methods can not distinguish whether the measured TPA is active or inactive because such assay methods monitor total TPA.
Labelling of the serine protease thrombin has been demonstrated by Paul Bock in an article entitled "Active Site Selective Labelling of Serine Proteases with Spectroscopic Probes Using Thioester Peptide Chloromethyl Ketones: Demonstration of Thrombin Labelling Using [(acetylthio)acetyl]-D-Phe-Pro-Arg-CH.sub.2 Cl". The subject article was originally printed in Biochemistry, 1988, 27, 6633. Although Bock describes a method for attaching ,fluorescent probes to thrombin, by means of covalent linkages, Bock does not describe or suggest a method for inhibiting the activity of plasminogen, a zymogen, so that the plasmin produced as a result of activating cleavage is catalytically inactive. The structure and function of plasmin and thrombin differ, the reactions which they catalyze differ, and thrombin is not a precursor, or zymogen, as plasminogen is.
Therefore, it is desirable to have an active site labelled plasminogen prepared in a manner which renders it an enzymatically or catalytically inactive enzyme upon conversion to plasmin. That is, a method which would allow direct determination of the action of two chain urokinase and single and two chain TPA, in an assay, without the complications associated with feedback reactions catalyzed by plasmin. This requires a method for making a labelled plasminogen which is enzymatically and catalytically inactive upon conversion to plasmin, and an assay method which utilizes the labelled plasminogen.